Host/vector system for expression of membrane proteins

ABSTRACT

A method of expressing proteins is disclosed. In a preferable embodiment, the method comprises placing a DNA sequence encoding a protein or peptide and expression vector containing a regulatable promoter expressible in  Rhodospirillum rubrum  and expressing the protein within a bacterial host, wherein the host has extra capacity for membrane formation and wherein the host is a member of the genus Rhodospirillum.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to Ser. No. 60/153,576, filedSep. 13, 1999. Ser. No. 60/153,576 is incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with United States government supportawarded by the following agencies: NIH GM57322. The United States hascertain rights in this invention.

BACKGROUND OF THE INVENTION

[0003]Rhodospirillum rubrum is a facultatively phototrophic purplenonsulfur bacterium. Under reduced oxygen concentration, this organismforms an intracytoplasmic membrane (ICM) that is the site of thephotosynthetic apparatus (Collins, M. L. P., and C. C. Remsen, Thepurple phototrophic bacteria, p. 49-77, In J. F. Stolz (ed.), Structureof Phototrophic Procaryotes. CRC Press, Boca Raton Fla., 1990; Crook, S.M., et al., J. Bacteriol. 167:89-95, 1986; Hessner, M. J., et al., J.Bacteriol. 173:5712-5722, 1991). This apparatus consists of thelight-harvesting antenna (LH) and the photochemical reaction center(RC). The pigment-binding proteins, the LH α and β and the RC-L and -M,are encoded by the puf operon while RC-H is encoded by puhA. Thenucleotide sequences of puhA and the puf operon have been determined inR. rubrum (Bélanger, G., et al., J. Biol. Chem. 263:7632-7638, 1988;Bérard, J., et al., J. Biol. Chem. 261:82-87, 1986; Bérard, J., and G.Gingras, Biochem. Cell Biol. 69:122-131, 1991) and related bacteria(Donohue, T. J., et al., J. Bacteriol. 168:953-961, 1986; Kiley, P. J.,et al., J. Bacteriol. 169:742-750, 1987; Michel, H., et al., EMBO J.5:1149-1158, 1986; Michel, H., et al., EMBO J. 4:1667-1672, 1985;Weissner, C., et al., J. Bacteriol. 172:2877-2887, 1990; Williams, J.C., et al., Proc. Natl. Acad. Sci. 81:7303-7307, 1984; Williams, J. C.,et al., Proc. Natl. Acad. Sci. 80:6505-6509, 1983; Youvan, D. C., etal., Proc. Natl. Acad. Sci. 81:189-192, 1984; Youvan, D. C., et al.,Cell 37:949-957, 1984).

[0004]R. rubrum may grow phototrophically under anaerobic lightconditions or by respiration under aerobic or anaerobic conditions inthe dark. Because R. rubrum is capable of growth under conditions forwhich the photosynthetic apparatus is not required, and because thephotosynthetic apparatus and the ICM may be induced by laboratorymanipulation of oxygen concentration, this is an excellent organism inwhich to study membrane formation (Collins, M. L. P., and C. C. Remsen,supra, 1990; Crook, S. M., et al., supra, 1986).

[0005] In previous studies, the puf region was cloned and interposonmutations within this region were constructed (Hessner, M. J., et al.,supra, 1991). R. rubrum P5, in which most of the puf genes were deleted,was shown to be incapable of phototrophic growth and ICM formation. P5was restored to phototrophic growth and ICM formation by complementationwith puf in trans (Hessner, M. J., et al., supra, 1991; Lee, I. Y., andM. L. P. Collins, Curr. Microbiol. 27:85-90, 1993). These results implythat in R. rubrum the puf gene products are required for ICM formation.These results differ from those obtained with a puf interposon mutant ofRhodobacter sphaeroides (Davis, J., et al., J. Bacteriol. 170:320-329,1988) which was phototrophically incompetent but was still capable ofICM formation (Kiley, P. J., and S. Kaplan, Microbiol. Rev. 52:50-69,1988). In the case of R. sphaeroides, the formation of ICM in theabsence of the puf products may be attributable to the presence of anaccessory light-harvesting component (LHII) encoded by puc (Hunter, C.N., et al., Biochem. 27:3459-3467, 1988). This implies that R. rubrum isa simpler model for studies of membrane formation.

[0006] Because the puf-encoded proteins are required for ICM formationin R. rubrum and because the RC is assembled from puf and puhA products,it is important to evaluate the role of puhA-encoded RC-H in RC assemblyand ICM formation in R. rubrum.

[0007] Cheng, et al., J. Bacteriol. 182(5):1200-1207, 2000 and YongjianS. Cheng, “Molecular Analysis of Biochemical Intracytoplasmic MembraneProteins,” PhD thesis, UW-Milwaukee, August, 1998 describe the cloning,mutation, and complementation of the puhA region of R. rubrum. (Both ofthese documents are incorporated herein by reference.) The presentapplication proposes a model for the preparation of proteins, preferablymembrane proteins.

SUMMARY OF THE INVENTION

[0008] In one embodiment, the present invention is a method ofexpressing protein comprising the steps of placing a DNA sequenceencoding a protein or peptide in an expression vector that contains aregulatable promoter expressible in Rhodospirillum rubrum and expressingthe protein within a bacterial host, wherein the host has extra capacityfor membrane formation and wherein the host is a member of the genusRhodospirillum.

[0009] In a preferred embodiment of the present invention, the proteinor peptide is a membrane protein or peptide and/or the protein orpeptide is a heterologous protein or peptide.

[0010] In another preferred form of the present invention, the host isRhodospirillum rubrum.

[0011] In another embodiment, the present invention is a proteinexpression system. In one embodiment, the protein expression systemencompasses a vector comprising a DNA molecule encoding the protein orpeptide in an expression vector containing a regulatable promoterexpressible in R. rubrum. The vector is contained within a host,preferably R. rubrum with extra capacity for membrane formation.

[0012] Other objects, advantages and features of the present inventionwill become apparent to one of skill in the art after review of thespecification, claims and drawings.

DETAILED DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0013]FIG. 1 is a diagram illustrating the construction of theexpression cassettes and the DNA sequence of the cassette of pREX1. ThePS fragment extends from position 92-429 and the T fragment extends fromposition 469-671. The multiple cloning site is from position 430-468.Sequences from position 1-91 and 672-857 are those of the vector. PM, PLand PG are not shown and include additional upstream sequences. Theexact lengths and positions of PS/PM, PL and PG are specified by thesequences of the primers reported herein and in Cheng thesis, supra.

[0014]FIG. 2 diagrams the construction of various expression vectors.PCR products PS, PM, PL and PG (not shown) from the various puh regionsare shown in position and size relative to the map of puh and flankingORFs.

[0015]FIG. 3 is a diagram of modular cloning of pPSpuhT or pPMpuhT. PCRprimers incorporated appropriate restriction sites to facilitate cloningof pPSpuhT or pPMpuhT. The promoter and terminator sequences were placedat the ends of the multiple cloning site to allow flexibility indesigning PCR primers for cloning genes inserted between the promoterand terminator. Mutated bases are indicated by the dotted boxes.

[0016]FIG. 4 is a set of absorbance spectra of R. rubrum H15complemented with pPSpuhT and pPMpuhT. Cells were grown phototropically(top of spectra) or semi-aerobically (bottom of spectra).

DESCRIPTION OF THE INVENTION

[0017] In one embodiment, the present invention is a host/vector systemfor expression of proteins. In preferable embodiments of the presentinvention, the protein is a heterologous protein and/or a membraneprotein. While numerous expression vector systems are commerciallyavailable, these vector systems generally cannot be applied to membraneproteins. Over-expression of membrane proteins is often toxic for thecell or results in the production of inclusion bodies in which theprotein is in a non-native structure.

[0018] By “membrane proteins” we mean proteins normally or naturallylocated in the cell membrane. Such proteins generally have one or moremembrane-spanning domains.

[0019] A host designed for the expression of membrane proteins in thepresent invention should have extra capacity to proliferate membranes toaccommodate the expressed protein. Extra capacity would avoid problemswith formation of inclusion bodies or lethality associated withover-expressed membrane proteins. By “extra capacity” we mean that thehost organism has the ability to make an intracytoplasmic membrane (ICM)but has a reduced ability to produce native membrane proteins.

[0020] The ability of a bacterium to make an ICM can be determined(assayed) by examining a sample with the electron microscope. ICM isknown to be made ordinarily by only three known groups ofbacteria—phototrophs (such as R. rubrum), methanotrophs andammonia-oxidizers. The latter two (especially ammonia-oxidizers) are notpreferred for molecular biology applications because of their growthrequirements.

[0021] The extra capacity for ICM formation by the mutants describedherein is due to mutation in the genes encoding the major membraneproteins—i.e., the proteins of the photochemical complexes. Because thebacteria retain the capacity to make ICM, they have “extra capacity.”

[0022] The host is a member of the genus Rhodospirillum and mostpreferably one of several mutants of Rhodospirillum rubrum. Preferably,the mutant hosts are defective in the production of puhA-encoded RC-H(Rhodospirillum rubrum H15) or puhA-encoded RC-H and puf-encoded LH-α,LH-β, RC-L and RC-M (Rhodospirillum rubrum H1). A puf knock-out mutant(Rhodospirillum rubrum P5 or P4) would also be suitable. (Note that P5mutant has a puf phenotype but still retains a partial pufB but pufALMis completely deleted.) Another puf mutant, R. rubrum P4 (described inJester, B., M S Thesis, University of Wisconsin at Milwaukee, May 1998,incorporated by reference herein), which is a puf knock-out but differsfrom P5 in that more genomic DNA is removed, is also suitable.

[0023] The basis for the mutational design described herein is that thehost's ability to produce its own native major membrane proteins hasbeen disrupted, thus providing the “extra capacity” to incorporateheterologous membrane proteins. For R. rubrum this means knocking ordisrupting out puh and/or puf. For other phototrophic bacteria (e.g.,Rhodobacter sphaeroides), it would be preferable to also knock out pucwhich encodes an additional photochemical component that is a majormembrane protein. Such an R. sphaeroides mutant has been constructed(see M. R. Jones, et al., Molec. Microb. 6:1173-1184, 1992).

[0024] While these R. rubrum mutants are impaired in ICM formation, theyretain the capacity to form an intracytoplasmic membrane in response tothe synthesis of membrane proteins, including heterologous membraneproteins. (Cheng, et al., supra, 2000, describes a comparison of theproperties of wild-type Rhodospirillum rubrum and mutated Rhodospirillumrubrum and describes the development of a suitable host for the presentinvention. (This article is incorporated by reference as if fully setforth herein.) In addition, Cheng, et al., supra, 2000, reports that thepuh promoter is contained within pH 3.6+. This promoter is incorporatedinto the expression vector described herein and is derepressed (i.e.,induced) by semi-aerobic conditions.

[0025] The R. rubrum system is advantageous, in part, because R. rubrumdoes not infect humans or animals and grows on a simple medium. Theintracytoplasmic membrane that houses the expressed protein may beseparated from the other particulate cellular material.

[0026] One may most easily obtain a suitable R. rubrum host byconstructing organisms analogous to P5 or H15. P5 may be reconstructedby following Hessner, et al., supra, 1991. H15 may be reconstructed byfollowing the procedure of Cheng, et al., supra, 2000.

[0027] An expression vector of the present invention should have thefollowing properties: (a) strong promoter; (b) regulated promoter; and(c) promoter regulated by a stimulus that is simple, inexpensive andnon-toxic. The parent plasmid used to construct the expression vectormust be capable of replication in a R. rubrum host. We have used IncPplasmids to construct the expression vector. However, IncQ plasmids alsoreplicate in R. rubrum, and one preferred embodiment of the presentinvention would be to move the cassette into an IncQ plasmid. Becausethese plasmids would be compatible in the host, this will make itpossible to simultaneously express two proteins. This embodiment couldbe applied to the synthesis of a membrane protein that is a heterodimer.Alternatively, it may be possible to use a single vector to expressoligomeric proteins that are co-transcribed on a single message.

[0028] The expression vectors preferably include an R. rubrum promoterwhich can be induced by reduction of oxygen. In addition, to being ableto replicate in the Rhodospirillum rubrum host, the expression vectormust have a promoter that is expressed strongly in this host.

[0029] Our development of a suitable expression vector is based on ourstudies of puh expression. The expression vector pREX1 (also known aspPST) is a construct in which cloning sites are located between promoterand terminator sequences.¹ These sequences are derived from the puhregion of pH 3.6±. The expression vectors pREX2 (also known as pPMT) andpREX3 (also known as pPLT, not yet built) contain longer portions of theR. rubrum sequence contained within pH 3.6−. The promoter can be inducedby reduction of oxygen tension. When the gene encoding a desiredprotein, such as a membrane protein, is cloned into this expressionvector and the vector is introduced into a suitable host, such asRhodospirillum rubrum H15 or Rhodospirillum rubrum H1, this protein canbe expressed by reducing the oxygen tension. This expression has beendemonstrated by the expression of Escherichia coli MaIF inRhodospirillum rubrum H15, as described below in the Examples.

[0030] The construct pH 3.6− has a strong promoter which results in thesynthesis of mRNA encoding the abundant protein PuhA. This promoter isregulated by oxygen and it can be derepressed by simple manipulationsapplicable to both lab scale and production scale. This avoids the useof chemical inducers which may be costly and/or toxic. NOMENCLATURE OFEXPRESSION VECTOR AND CLONES U.S. Ser. No. This application 60/153,576Cheng thesis pREX1, pPST pPST pPST pREX2, pPMT pPLT pPLT pPREX3, pPLT —— pPSpuhT pPSpuhT pPSpuhT pPMpuhT pPLpuhT pPLpuhT pPSpuh88T — pPSHD1T

[0031]FIG. 1 is a diagram illustrating the construction of theexpression cassettes and the DNA sequence of the cassette of pREX1. TheDNA sequence includes a small portion of the vector sequence that couldbe used in subcloning a cassette into various vectors. The sequence ofthe puh region was reported in the literature. (Bérard, J., et al., J.Biol. Chem. 264:10897-10903, 1989 and Bérard, J. and Gingras, G,Biochem. Cell Biol. 69:122-131, 1991).

[0032] We envision the construction of pPLT and pPGT, alternatives withmore upstream sequence, as follows:

[0033] Longer promoter fragments will be amplified using PF3EcoRI andPF4SacI. (See Table 1 below.) pRK415 will be used as a platform. The PGfragment amplified using PF4SacI contains all of the sequence upstreamof puhA in pH 3.6− and extends into the additional sequence reported inCheng, et al., supra, 2000. TABLE 1 Amplification Primers TFSphIGTAATTGGGGGCATGCCACATGGATGA TRHindIII CGGCGGTCAGAAGCTTGGGCAGCGGATPF3EcoRI GCAACCAAGGAATTCCCGCTGGGTCGT PRSacI GAGGGTGACGAGCTCTCCTGGGAACTCPRSacI ATGACCAGTTGAGCTCCCATCCAGCCGCTTGG

[0034] pPLT will be constructed with a similar strategy for pPST andpPMT. In brief:

[0035] 1. One would PCR amplify PL product with PF3EcoRI and PRSacI

[0036] 2. Digest with EcoRI and SacI or SstI

[0037] 3. Digest pRK415 Eco and SacI or SstI

[0038] 4. Ligate PL fragment into digested vector to construct pPL;transform E. coli.

[0039] 5. PCR amplify T fragment with TRHindIII and TFSphI

[0040] 6. Digest T PCR product with SphI and HindIII

[0041] 7. Digest pPL with SphI and HindIII

[0042] 8. Ligate T fragment into digested pPL

[0043] The strategy for pPGT is similar:

[0044] 1. PCR amplify T fragment with TR and TF

[0045] 2. Digest T PCR product with SphI and HindIII

[0046] 3. Digest pRK415 with SphI and HindIII

[0047] 4. Ligate T fragment into digested pRK415 to form pRKT

[0048] 5. PCR amplify PG product with Pf4SacI and PRSacI

[0049] 6. Digest pRKT with SacI

[0050] 7. Ligate PG fragment into SacI-digested pRKT

[0051] 8. Transform E. coli and screen transformants to identify thosewith the PG sequence in the correct orientation.

[0052] To build a construct that expresses a desired protein, thefollowing steps would preferably be used:

[0053] 1. Amplify by PCR the structural gene encoding the protein. Forthis purpose, the PCR primers should incorporate a restriction siteavailable in the polycloning site such that the amplified product can beinserted in the correct orientation. The sites must be in the multiplecloning site and not present in the parent plasmid used to construct theexpression vector nor in the structural gene being cloned. Thepromoter/MCS/terminator sequences (FIG. 1) can be subcloned intodifferent parent plasmids and this will affect the sites available forcloning. Primers may incorporate an optimized ribosomal binding site. Toclone a partial sequence, initiation and termination codons as well as aribosomal binding site preferably should be engineered into the primers.To clone eukaryotic proteins, the template DNA preferably should be cDNAin order to avoid introns.

[0054] 2. Trim the purified PCR product with the appropriate restrictionenzymes. It would be possible to use the same restriction site on bothprimers. However, under these conditions, this will not be “directionalcloning” and it will be necessary to screen recombinants (restrictionanalysis) to identify those in the proper orientation.

[0055] 3. Ligate the trimmed fragment into the expression vectordigested with the appropriate enzymes. In addition to pJB3Cm6 (mentionedin the Examples), other vectors that could preferably be used as aplatform for the expression fragment are pSUP104, pJRD215 and pKT210.(See [for pPSUP104]: Priefer, U. B., et al., J. Bacteriol. 163:324-330,1985; [for pJRD215]: Davison, J., et al., Gene 51:275-280, 1987; [forpKT210]: Priefer, U. B., et al., J. Bacteriol. 163:324-330, 1985.)

[0056] 4. Transform a suitable E. coli strain (such as S17-1) with theconstruct.

[0057] 5. Conjugate the construct into the R. rubrum host.Alternatively, R. rubrum can be electroporated or transformed. Selectfor transconjugants with the appropriate antibiotic. The appropriateantibiotic will be determined by the selection markers on the parentplasmid and the host.

[0058] 6. Culture transconjugant under aerobic conditions.

[0059] 7. Reduce oxygen tension to derepress cloned gene under controlof puh promoter.

[0060] There are numerous potential applications for the host/vectorsystem of the present invention. For example, biotechnologyinvestigators could use the system in basic science applicationsconcerning the numerous putative genes that have been identified andcontinue to be identified by genome sequencing. Immunological andbiochemical approaches to understanding the role of these genes inhealthy and diseased cells will require expression of the genes. Thisnew host/vector system is uniquely suited for expression of membraneproteins.

[0061] Physical analysis (e.g. X-ray crystallography) requires milligramquantities of pure protein. This requirement has limited the applicationof this type of analysis to only a few membrane proteins, largely thosethat are highly expressed in their natural host. Expression of membraneproteins in the new host/vector system would extend this approach tomany membrane proteins of importance. This would include, for example,receptors which play a role in intercellular communication in the immuneresponse, neuroendocrine function, viral infection, and other importantphysiological activities.

[0062] Many immunoprotective antigens of viruses, bacteria and otherinfectious agents are membrane proteins. One of the most importantpotential applications of the present invention would be to producevaccines. This host/vector system could be used for the production ofnew, improved or more cost-effective subunit vaccines. Among thepotential advantages of this system are the following: (1) vaccine wouldnot be infectious, (2) large scale production should be efficient, (3)proteins from difficult-to-cultivate pathogens could be expressedprovided that sufficient sequence information is available to design PCRprimers, and (4) the protein should be assembled in the membrane in itsnative (antigenic) state.

[0063] One potential obstacle is that the expressed protein would not bemodified as in the native host. In the case of modification byproteolytic cleavage, this obstacle may be overcome by engineering atruncated protein. Viral proteins that are normally glycosylated wouldnot be modified when produced in this R. rubrum host/vector system.However, non-glycosylated proteins may stimulate the production ofprotective antibody as has been found to be the case with therecombinant vaccine now in use for hepatitis B.

EXAMPLES

[0064] Vectors designed for the expression of membrane proteins in R.rubrum H15 were constructed. These constructs were based on pJB3Cm6because this vector is small and fully sequenced. See, Blatny, J. M., etal., Appl. Environ. Microb. 63:370-379, 1997. Also note, the sequence ofthe puh region is reported in Bérard, J., et al., supra, 1989 andBérard, J. and Gingras, G, supra, 1991. Expression of cloned genes willbe driven by puh expression sequences contained within pH 3.6−. Becauseof uncertainty in the length of sequence required for oxygen regulatedexpression, four putative puh promoter sequences of differing length(designated S and M done, L and G in progress) will be amplified by PCRand cloned into this vector to form pPS, pPM, pPL and pPG (FIG. 2). Theputative puh terminator sequence was or will be cloned into each ofthese to form pPST, pPMT, pPLT and pPGT (FIG. 2). These sequences flankmultiple cloning sites into which genes intended for expression may becloned.

[0065] To test the capacity to express genes, the homologous gene puhAwas used as a reporter. PuhA was amplified by PCR using primers thatincorporated restriction sites for SacI and SphI. The PCR product andthe vectors were treated with these enzymes and the PCR fragments wereligated to the vectors to form pPSpuhT and pPMpuhT (FIGS. 2 and 3).These constructs were used to transform Escherichia coli S17-1 that wasin turn conjugated to the puh knock-out strain R. rubrum H15. BothpPSpuhT and pPMpuhT restored phototrophic growth and photopigmentcontent to H15 and the photochemical reaction center was detected byspectroscopy in cells incubated under phototrophic or semi-aerobicconditions (FIG. 4). The vector controls (pPST and pPMT) did not restorethe phenotype. These results suggest that both expression vectors willfunction in R. rubrum for puhA expression.

[0066] To test the expression of a heterologous protein, MaIF wasamplified by PCR using total DNA from E. coli MC4100 as a template. ThisPCR product was cloned into pPST to form pPSMaIFT (FIG. 2). This plasmidwas transferred to R. rubrum H15 by conjugation and H15(pPSMaIFT) wasincubated under semi-aerobic conditions to evaluate expression of maIF.Membranes were prepared from H15(pPSMaIFT) and H15(pPSpuhT); the latterserved as a negative control. When analyzed by SDS-PAGE, MaIF was notdetected (not shown). When analyzed by immunoblot, MaIF was detected inmembranes prepared from H15(pPSMaIFT) but not H15(pPSpuhT). Theseresults indicate that while expression of MaIF was achieved,hyperexpression was not.

Use of pREX to Express a Truncated Protein

[0067] The expression of puhA from pREX (aka pPST) provided theopportunity to evaluate a truncated puhA. The reverse primer 88R (below)was designed to incorporate a termination codon as well as a restrictionsite. The fragment amplified with HF and 88R, which encodes the first 88amino acids of RC-H, was cloned into pREX to form pREXpuh88. Thephenotype of H15 complemented with this construct was evaluated. Thistruncated puhA restored the photopigment content of the membrane to alevel equivalent to that obtained with pREXpuh which is reflected in thespectrum. The spectrum also shows a peak at 800 nm indicative of thephotochemical reaction center (RC). This RC formed with a truncated RC-His functional because H15 (pREXpuh88) is capable of phototrophic growth.The ability to grow phototrophically was lost when H15 was cured ofpREXpuh88. res. primer sequence 5-3 site HF GTTCCCAGGAgAGCtCGTCACCCTCAGSacl 88R GCGCGGTGCGCaTGcCTTaGATCGCGACGGCATC³ Sphl

[0068]

1 8 1 857 DNA Artificial Sequence Description of ArtificialSequenceexpression vector 1 cagctggcga aagggggatg tgctgcaagg cgattaagttgggtaacgcc agggttttcc 60 cagtcacgac gttgtaaaac gacggccagt gaattcggtgggcacgctga ccgcggcgat 120 ggcgctggcc gatgaaacgg tcagcggaat ggcgctcggcgcttggggcg ccgtgcaggc 180 caccgcgacc ggcgcggccg ttgcccttgg cggcggcttgcgcgatggcg tttcctcgtt 240 ggcggcccat ggcctgctcg gcgaggcctt aaccacggcccatacgggct atggtttcgt 300 ttatctggta gaagttgttt tgttatttac aaccttggccatcatcggcc cgctggttcg 360 tacggccgga caccgcgcgt cccagtcttc ggaaggacgtttcggtttgg ccgagttccc 420 aggagagctc ggtacccggg gatcctctag agtcgacctgcaggcatgcc acatggatga 480 gtacgattcc gaaccgatcc gtggactgcc tgcggatctgccgccgggcg aattcatcct 540 gtggcagggc gcgccgacac ggcgcgccct tgccctccgggtgtttcaca ttcggctgat 600 cgcgctttat ttcgcgattc tggtggcgtg gaacgtggcctcggctttgt atgacggcca 660 tccgctgccc aagcttggcg taatcatggt catagctgtttcctgtgtga aattgttatc 720 cgctcacaat tccacacaac atacgagccg gaagcataaagtgtaaagcc tggggtgcct 780 aatgagtgag ctaactcaca ttaattgcgt tgcgctcactgcccgctttc cagtcgggaa 840 acctgtcgtg ccagctg 857 2 27 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 2 gtaattgggggcatgccaca tggatga 27 3 27 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 3 cggcggtcag aagcttgggc agcggat 27 427 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 4 gcaaccaagg aattcccgct gggtcgt 27 5 27 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 5 gagggtgacgagctctcctg ggaactc 27 6 32 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 6 atgaccagtt gagctcccat ccagccgcttgg 32 7 27 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 7 gttcccagga gagctcgtca ccctcag 27 8 34 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 8 gcgcggtgcgcatgccttag atcgcgacgg catc 34

We claim:
 1. A method of expressing proteins comprising the steps of (a)placing a DNA sequence encoding a protein or peptide in an expressionvector comprising a regulatable promoter expressible in Rhodospirillumrubrum, and (b) expressing the protein within a bacterial host, whereinthe host has extra capacity for membrane formation and wherein the hostis a member of the genus Rhodospitillum.
 2. The method of claim 1wherein the protein or peptide is a membrane protein or peptide.
 3. Themethod of claim 1 wherein the protein or peptide is a heterologousprotein or peptide.
 4. The method of claim 2 wherein the protein is aheterologous protein.
 5. The method of claim 1 wherein the host isRhodospirillum rubrum.
 6. The method of claim 5 wherein the host isdefective in the production of puhA-encoded RC-H.
 7. The method of claim6 wherein the host is Rhodospirillum rubrum H15.
 8. The method of claim1 wherein the host is defective in puhA-encoded RC-H and puf-encodedLH-α, LH-β, RC-L and RC-M.
 9. The method of claim 8 wherein the host isRhodospirillum rubrum H1.
 10. The method of claim 1 wherein the host isa puf knock-out mutant.
 11. The method of claim 10 wherein the host isRhodospirillum rubrum P5 or Rhodospirillum rubrum P4.
 12. The method ofclaim 1 wherein the expression vector comprises a promoter that can beinduced by reduction of oxygen tension.
 13. A protein expression systemcomprising: a vector comprising a DNA molecule encoding a protein orpeptide in an expression vector comprising a regulatable promoterexpressible in R. rubrum, wherein the vector is contained within a hostof the genus Rhodospirillum and wherein the host has extra capacity formembrane formation.
 14. The protein expression system of claim 13wherein the protein or peptide is a heterologous protein or peptide. 15.The protein expression system of claim 13 wherein the protein or peptideis a membrane protein or peptide.
 16. The protein expression system ofclaim 14 wherein the protein or peptide is a membrane protein orpeptide.
 17. The protein expression system of claim 13 wherein the hostis Rhodospirillum rubrum H15.
 18. The protein expression system of claim13 wherein the host is defective in the production of puhA-encodedRCH-H.
 19. The protein expression system of claim 13 wherein the host isR. rubrum.
 20. The protein expression system of claim 19 wherein thehost is defective in the puhA-encoded RCH-H and puf-encoded LH-α, LH-β,RC-L and RC-M.
 21. The protein expression system of claim 13 wherein thehost is Rhodospirillum rubrum H1.
 22. The protein expression system ofclaim 1 wherein the host is a puf knock-out mutant.